Parenteral amino acid solution



Patented Dec. 8, 1953 PARENTERAL AMINO ACIDSOLUTI ON Eugene E. Howe, Bound Brook, N. .L, assignor to Merck & 00., Inc., Rahway, N. J., a corporation of New Jersey No Drawing. Application June 19, 1948,

Serial No. 34,151

6 Claims. (01.167-58) This invention relates to amino acid mixtures and particularly to amino acidsolutions of the type adapted for parenteral injection. More particularly, the invention relates to an improved method of adjusting the pH of amino acid mixtures which results in a drastic reduction in toxic eiTects and objectionable side reactions when such solutions are parenterally administered, and to the new amino acid mixtures thus obtained.

In the past amino acid solutions for parenteral injection have generally been either acid or enzymatic protein hydrolyzates. In these hy drolyzates essential amino acids including both monoamino monocarboxylic acids and the basic amino acids l-arginine, Z-histidine and Z-lysine are present, and the basicity of the mixture is essentially neutralized by dicarboxylic acids such as glutamic and aspartic acids which are also present in the hydrolyzates. It has been found, however, that the dicarboxylic acids are largely responsible for the nausea and vomiting generally experienced in the parenteral administration of protein hydrolyzates.

In my pending joint application Howe and Tishler Serial No. 637,498, filed December 27, 1945, now United States Patent No. 2,457,820, issued January 4, 1949, there is disclosed a new procedure for fractionating protein acid hydrolyzates to separate monoamino monocarboxylic acid and basic amino acid fractions and recombining such fraction with added amounts of glycine and racemic essential amino acids including tryptophane to form improved amino acid solutions containing the ten natural essential amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane and valine) in the physiologically active L-form'and characterized as being free of dicarboxylic acids and containing not more than 5% of theiessential amino acids in the inactive D-form. In these new amino acid solutions the basicity has heretofore been neutralized with hydrochloric acid.

According to the procedure disclosed in said application Serial No. 637,498, a protein acid 'hydrolyzate is extracted with butanol to remove a fraction of monoamino monocarboxylic acids and the residual hydrolyzate is then treated with an adsorbent material which preferentially adsorbs basic amino acids. The adsorbed basic amino acids are then eluted with mineral'acid, thus effecting a neutralization of the basic amino acids. In accordance with an improved procedure more specifically disclosed in my co-pending joint application Howe and Tishler Serial No. 764,569, filed July 29, 1947, now United States Patent No; 2,480,654 issued August 30, 1949, the basic amino acids are eluted from the adsorbent material with 2 about 20% aqueous ammonia and the eluate is concentrated to remove excess ammonia. When following this procedure the M. A. fraction and the basic fraction are combined and adjusted to a pH of about 6.5-7.0 by addition of hydrochloric acid. While amino acid solutions neutralized in the manner above described appeared initially to have no detrimental effects, it has been found that presence of hydrochloric acid in the amounts required for the neutralization of the basic amino acids tends at least in some instances to combine with sodium or ammonium ions of the blood and to be excreted in the urine, causing the loss of fixed base and CO2 combining power of the blood.

' It has now been discovered in accordance with the present invention that the disadvantages introduced by neutralizing with hydrochloric acid can be wholly avoided and distinctly superior amino acid mixtures can be prepared by employing as the neutralizing agent a physiologically innocuous organic acid.

By physiologically innocuous organic acid is meant any of the group of organic acids which are assimilated and utilized by the body, or organic acids which are formed in the metabolism of foodstuffs and thus occur normally in the body, which when introduced into the bloodstream in high concentration produces no untoward side reactions such as nausea, vomiting, loss of calcium, etc.

Various organic acids are suitable for this purpose including acetic, lactic, pyruvic, and pyrrolidone carboxylic acids. It is considered,,however, that lactic acid and pyrrolidone carboxylic acid are the acids best suited for neutralizing the basic amino acids.

It should be noted that the use of physiologically innocuous organic acids in the neutralization not only avoids the objectionable effect on CO2 combining power of the blood after infusion, but also avoids the objectionable nausea and vomiting produced by glutamic and aspartic acids. In addition, the physiologically innocuous acids increase the caloric content of the amino acid solutions as compared with solutions neutralized with hydrochloric acid.

The following examples show how the process of the present invention can be carried out, but it is to be understood that these examples are given by way of illustration'and not of limitation.

EXAMPLE 1 Preferred procedure for fractionating protein acid hydrolyzate One kgm. of casein (ash-free moisture-free basis) is placed in a 5 l. flask with 3300 cc. of

The residual syrup is dissolved in about "15' liters of condensed water and .filteredthrougha thin bed of charcoal to remove insoluble humin. The filtrate (pl-I 0.5 to.1.0)-:is;..then passed through a kilogram of Amberlite1=IR+4B .(anionexchange polyamine resin prepared according to Example 4 of U. S. Patent No..2,402,384 issued June 18, 1946, to J. W. Eastes), arranged-in .a column about 3 ft. long. The Amberlite is prepared for use by treating with hydrochloric Y acid, then treating with-% sodium hydroxide, andwashingtopH78.. '1. The filtrate is passed through the column at a rate .of about 200 cc/min. and the column is washedwith 2--3 l. of water at the. same-rate. The e'filuent and washings are collected. until the pH reaches 5, and are then agitated for. EEO-#15. minJWith about 100 gm. of charcoal. Thecharcoal'is then filtered ofi and the filtrate 'is-concentrated to about 51. At this point, the nitrogencontent is about'2.8 gm./100 cc. which represents. a loss of only about 3% of that'in the original casein. The solution is chilled to Oi-5 C. for about '8 hours causing precipitation of tyrosine which is removed by filtration.

f The hydrolyzate is then placed in a semi-continuous liquid extractor and extracted with about volumes of butanol'at such a pressure (approximately 60 .mm. Hg) that the temperature does not exceed about 65 C. The time'for extraction is about 36' hours.

The butanol-extracted hydrolyzate is now passed through the column, the basic amino acids being adsorbed on the resin. It is found that'approximately 2000 gm. of resin to 1 kilo of original caseinwill completely adsorb without breakthrough the basic amino acids present in the extracted hydrolyzate.

The column is. then eluted with 6 liters of 20% ammonia. The collected eluate is concentrated .to about.'1;'75.litersi-and on analysis is found to containabout gm. of arginine, 25 gm. of histidine and 70 gm. of lysine. This is approximately 90% of. the basic amino acids present in .thehydrolyzate from one kilo of casein.

The M. A. fraction and the basic fraction thus prepared-are ready for use in preparing solutions containing balanced mixtures of essential amino: acids. The following examples show how such mixtures can be prepared.

. EXAMPLE 2 A casein acid hydrolyzate was treated in accordance with the procedure described in'Example 1. to prepare separate monoamino monocarboxylic acid and basic amino acidfractions.

55 gms. of the monoamino carboxylic acid fraction was dissolved in 600 cc. of distille'dwasolution was filtered through a thin' layer of The vbutanol containing the precipitated M. A. fraction, .i. e.

monamino monocarboxylic acids, is chilled to 0-5 C. for about 8 hours, then filtered; and the precipitate is 'washed with two 250.:cc. portions of dry butanol and then with two 1250 cc. .por-

butanol and passed through a 6 it. column containing 2000 gm. of Zeokarb NH4 oriAmberlite IR-IOO (moisture-free basis) at the rate of about (This product contains essential monocharcoal and was neutralized to pH 7 with 7.0- gm.of lactic acid. 22.6 gm. of glycine was added to the effluent solution and its pH was adjusted to 7.4.

The volume was adjusted to one liter "after which 10:0 gm. of Darco was added and the solution was filteredinto a pyrogen free flask. Oxygen was expelled by passing nitrogen into the solution and 1.8 gm. of dZ-tryptophane was added and dissolved by shaking. To this 0.5 gm. of sodium bisulfite was added, and the solution was sterilized by filtration and transferred sterilely to 500 cc. serum'bottles.

EXAMPLE 3 The procedure of Example'2 was repeated us ing glacial acetic acid in place of lactic acid to effect neutralization. Approximately gms. of glacial acetic acid was required to neutralize :1 to .pH'7. trated to about 4 liters for removaluof dissolved 300' cc. per min. The ,columnis prepared: as

follows: Fill an S'ft. lengthof, mm. :glass' tubing half full of water and introduce 2000- gm. ofZeokarb H (10% moisture) or 2000- gm.- -of Amberlite -IR',-100. (Zeokarb H is ar-sulfonated coal, and Amberlite IR100 is a modified phenol formaldehyde methylol sulfonic acid typeresin.) The column is washed with condensed water until the wash is relatively free of color. Six liters of 20% aqueousammonia is then passed through the column at 300 cc. per min., and the column is then washed with about 20 liters of condensed water. The column is now ready for treatment of the basic amino acid fraction, although final conditioning of the column is efiected 'b'y the initial'run of basic amino acids. Thus the first batch of basic fraction treated the. column should be tested separately for tolerance and toxicity.

EXAMPLE 4 1'I'he procedure of Example 2 was repeated using 'l-pyrrolidone carboxylic acid in place of lactic; acid to effect neutralization. Approximately. 5 :gms. of l-pyrrolidone carboxylic'acid was required to neutralize to pH 7.

- -EXAM'PLE "5 "The procedure of Example-2 was repeated us ing pyruvic acid in place of lactic acid to efiect neutralization. Approximately 5 gms. of pyruvic acid was required to neutralize to .pH '7.

It will be understood that in the foregoing pro ccdures solutions can'be neutralized to any pI-I suitable for infusion into humans. Thus the pH may be-adjusted by addition of a physiologically innocuous organicacid to any desired point within a range of about pH 4.5 to 8.

A number of clinical tests conducted by difier ent investigators using amino acid preparations neutralized as described in the foregoing exampleshave shown that these amino acidmix tures do not produce changes in the CO2 combining power of the blood characteristic of acidosis, and that these mixtures are better tolerated in man than similar mixtures neutralized with hydrochloric acid which were previously employed.

Various changes and modifications in the foregoing procedure will occur to those versed in the art, and to the extent that such changes and modifications fall within the purview of the appended claims it is to be understood that they constitute part of my invention.

'1 claim:

1. A parenteral solution of amino acids containing not more than 5% of the essential amino acids in the inactive D-form, solute components of said solution being free of dicarboxylic acids and comprising the natural essential amino acids, leucine, isoleucine, phenylalanine, valine, methionine, threonine, arginine, histidine, and lysine, racemic amino acids including tryptophane, the non-essential amino acid glycine, and an amount of a physiologically innocuous organic acid sufficient to neutralize the basicity of the basic amino acids arginine, histidine, and lysine.

2. A parenteral solution for amino acids containing not more than 5% of the essential amino acids in the inactive D-form, solute components of said solution being free of dicarboxylic acids and comprising the natural essential amino acids, leucine, isoleucine, phenylalanine, valine, methionine, threonine, arginine, histidine, and lysine, racemic amino acids including tryptophane, the non-essential amino acid glycine, and an amount of lactic acid sufficient to neutralize the basicity of the basic amino acids arginine, histidine, and lysine.

3. A parenteral solution for amino acids containing not more than 5% of the essential amino acids in the inactive D-form, solute components of said solution being free of dicarboxylic acids and comprising the natural essential amino acids, leucine, isoleucine, phenylalanine, valine, methionine, threonine, arginine, histidine, and lysine, racemic amino acids including tryptophane, the non-essential amino acid glycine, and an amount of pyrrolidone carboxylic acid suflicient to neutralize the basicity of the basic amino acids arginine, histidine, and lysine.

4. A parenteral solution for amino acids containing not more than 5% of the essential amino acids in the inactive D-form, solute components of said solution being free of dicarboxylic acids and comprising the natural essential amino acids, leucine, isoleucine, phenylalanine, valine, methionine, threonine, arginine, histidine, and lysine,

racemic amino acids including tryptophane, the non-essential amino acid glycine, and an amount of pyruvic acid sufiicient to neutralize the basicity of the basic amino acids arginine, histidine, and lysine.

5. A parenteral solution for amino acids containing not more than 5% of the essential amino acids in the inactive D-form, solute components of said solution being free of dicarboxylic acid and comprising the natural essential amino acids,

leucine, isoleucine, phenylalanine, valine, methionine, threonine, arginine, histidine, and lysine, racemic amino acids including tryptophane, the non-essential amino acid glycine, and an amount of acetic acid sufiicient to neutralize the basicity of the basic amino acids arginine, histidine, and lysine.

6. A parenteral solution of amino acids containing not more than5% of the essential amino acids in the inactive D-form, solute components of said solution being free of dicarboxylic acids and. comprising the natural essential amino acids, leucine, isoleucine, phenylalanine, valine, methionine, threonine, arginine, histidine, and lysine, racemic amino acids including tryptophane, the non-essential amino acid glycine, and a physiologically innocuous organic acid selected from the group consisting of lactic acid, acetic acid, pyruvic acid and pyrrolidone carboxylic acid in an amount suflicient to neutralize the basicity of the basic amino acids, arginine, histidine and lysine.

EUGENE E. HOWE.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 1,489,181 Weidner Apr. 1, 1924 2,101,867 Miller Dec. 14, 1937 2,416,956 Sahyun Mar. 4, 1947 2,457,117 Bernardi Dec. 28, 1948 2,457,820 Howe et al. Jan. 4, 1949 2,462,597 Block Feb. ,22, 1949 OTHER REFERENCES Hartmann, American Journal Pharm., November 1934, pages 424 to 435.

Madden et al., Journal of Experimental Medicine, volume 79, June 1, 1944, pages 607 to 624, page 622 relied upon.

Sahyun, Outline of the Amino Acids and Proteins, Reinhold Publishing Company, New York, 1944, page 128.

Physicians Bulletin, June 1947, page 81.

4Drug' and Cosmetic Industry, August 1945, page 2 0. 

1. A PARENTERAL SOLUTION OF AMINO ACISD CONTAINING NOT MORE THAN 5% OF THE ESSENTIAL AMINO ACIDS IN THE INACTIVE D-FORM, SOLUTE COMPONENTS OF SAID SOLUTIONS BEING FREE OF DICARBOXYLIC ACIDS AND COMPRISING THE NATURAL ESSENTIAL AMINO ACIDS, LEUCINE, ISOLEUCINE, PHENYLALANINE, VALINE, METHIONINE, THERONINE, ARGININE, HISTIDINE, AND LYSINE, RECEMIC AMINO ACIDS INCLUDING TRYPOTOPHANE, THE NON-ESSENTIAL AMONO ACID GLYCINE, AND AN AMOUNT OF A PHYSIOLOGICALLY INNOCUOUS ORGANIC ACID SUFFICIENT TO NEUTRALIZE THE BASICITY OF THE BASIC AMINO ACIDS ARGININE, HISTIDINE, AND LYSINE. 